Histone Modification Mechanisms for Control of Gene Expression
Histones are proteins found in the nuclei of eukaryotic cells that are used by cells to package genomic DNA into higher ordered structures called nucleosomes. The nucleosome comprises approximately 146-147 base pairs of DNA wrapped around the cylindrical core of an eight histone protein complex. The nucleosome forms the fundamental repeating unit of eukaryotic chromatin, and is organized via folding patterns of chains of nucleosomes to form higher ordered structures, and ultimately to form a chromosome.
Several families of histone proteins exist, including H1/H5, H2A, H2B, H3 and H4. Histones H2A, H2B, H3 and H4 are core histones. H1 and H5 are linker histones. Histones and DNA interact with each other in various ways, including hydrogen bonds, nonpolar interactions, salt bridges, and non-specific minor groove insertion. Many of these interactions are between the histone and the backbone repeating unit of DNA. For example, the core histone proteins, H2A, H2B, H3 and H4 are rich in lysine and arginine, which are amino acids with basic side chains. The positive charges of those basic side chains can effectively neutralize the negatively charged DNA backbone. The numerous interactions of histone proteins with the DNA backbone partially explain why almost all sequences of DNA can be bound to a histone octamer to form a nucleosome.
In addition to the core and linker histones, several histone variants are also known to exist, such as H2AZ, H2AX, and H3.3. The variant histones differ slightly in amino acid sequence from the core and linker histones. The nucleosomes that incorporate histone variants may differ in stability from those that only contain the core histones, and may be expressed at different times of organism development or cell cycle to accommodate different rates of DNA transcription or DNA replication. Incorporation of many histone variants has been associated with an altered chromatin structure.
Each of the core histone proteins also has a “tail” region which extends out from the histone-DNA core complex. The histone tails may be post-translationally modified. For example, the tails may be modified by lysine and arginine methylation, lysine acetylation, serine, threonine and tyrosine phosphorylation, and lysine ubiquitination and sumoylation. [Jenuwein, T. and Allis, C D, Translating the histone code, Sci. 293: 1074-80 (2001).] Such modifications can change the local charge of chromatin causing a more “closed” or more “open” state. Modification of the histone tails contributes to the epigenetic regulation of various nuclear processes.
Post-translational modifications to histones may be made that directly or indirectly alter the DNA-histone interaction. Common modifications to histones include acetylation, methylation, ubiquitination, and phosphorylation. Some modifications, such as acetylation or phosphorylation, can directly affect the charge of the histone and dramatically affect nucleosome dynamics. Various patterns of post-translational modifications can modulate the DNA-histone structure either by changing the affinity of the DNA for the histone complex, or by recruiting other proteins that alter the affinity of DNA for the histone complex.
Post-translational modification of histones provides a mechanism whereby a eukaryotic cell can modulate nucleosome and higher order chromatin structure to spatially and temporally control gene expression. A high level of control over gene expression is required for a cell to coordinate complex nuclear processes such as DNA replication, DNA repair, and transcription. The local and specific modulation of chromatin via histone modification is a fundamental mechanism that eukaryotic cells have evolved to achieve the requisite level of control over DNA processes.
The assembly of DNA into nucleosomes and further packing into higher-ordered chromatin forms a restrictive environment for various nuclear processes such as DNA replication, DNA repair, and transcription. This is so because such packing prevents various transcription, replication, and recombination factors from having access to the DNA.
A variety of chromatin remodeling factors modulate nucleosome and chromatic structure using the energy stored in molecules of ATP. For example, remodeling factors comprise SNF2 (Sucrose Non-Fermentable)-like family of the DEAD/Helicase Superfamily2 (HSF2) DNA-stimulated ATP-ases. The hSWI/SNF (SWItch/Sucrose Non-Fermentable) multisubunit complex, which is highly conserved, contains hBrahma (hBRM) or Brahma-related gene 1 (BRG1) ATPases that alter histone-DNA interactions. This in turn allows general transcription factor access to promoter regions of DNA. Thus, certain remodeling complexes may be targeted to promoters by way of interaction with sequence specific transcription factors. [See http://www.biocarta.com/pathfiles/h_hSWI-SNFpathway.asp citing Narlikar G J et al., Cooperation between complexes that regulate chromatin structure and transcription. Cell. 2002 108(4):475-87; Vignali M et al. ATP-dependent chromatin-remodeling complexes. Mol Cell Biol. 2000 (6):1899-910]
Some of the enzymes responsible for modification of histones can also modify proteins involved in other cellular processes. For example, the histone H3K4 methyltransferase SETT/9 catalyzes the methylation of various non-histone proteins, including DNMT1, p53, yes-associated protein (Yap), SUV39H1, and nuclear hormone estrogen receptor alpha (ERα), Zhang, G. and Pradhan, S., Mammalian Epigenetic Mechanisms, IUBMB Life 66(4): 240-256 (2014).
Histone Acetylation
One dynamic post translational modification to histones is the acetylation of the ε-amino group of lysine, which is regulated by opposed activities of lysine acetyltransferases (KATs) and histone deacetylases (HDACs). [Delcluve, G P, et al, Role of histone deacetylases in epigenetic regulation: emerging paradigms from studies with inhibitors, Clinical Epigenetics 4: 5 (2012).]
Acetylation of histone proteins modulates chromatin structure involved in the replication, repair, silencing and transcription of DNA. [Id; citing Groth, A., et al., “Chromatin Challenges during DNA replication and repair,” Cell 128: 721-33 (2007); Shahbazian, M D et al., “Functions of site-specific histone acetylation and deacetylation,” Ann. Rev. Biochem. 76: 75-100 (2007)]. Hyper acetylation is associated with a decondensed chromatin structure. [Id; citing Shahbazian, M D et al., Functions of site-specific histone acetylation and deacetylation, Ann. Rev. Biochem. 76: 75-100 (2007); Tse, C., et al, Disruption of higher order folding by core histone acetylation dramatically enhances transcription of nucleosomal arrays by RNA polymerase III, Molec. Cell Biol. 18: 4629-38 (1998); Wang, X, et al, Effects of histone acetylation on the solubility and folding of the chromatin fiber, J. Biol. Chem. 276: 12764-68 (2001); Shogren, K M, et al, Histone H4-K16 acetylation controls chromatin structure and protein interactions, Science 311: 844-847 (2006); Davie, J R, et al, Nuclear Organization and chromatin dynamics: Sp1, Sp3, and histone deacetylases, Adv. Enzyme Regul. 48: 189-238 (2008)]. Bromodomain modules of certain proteins, and sometimes lysine (K)—acetyl transferases (KATs), recognize acetylated sites on core histone proteins as part of the chromatin-remodeling mechanism involved in the activation of transcription. [Id; citing Lee, K K, Workman, J L, Histone acetyltransferase complexes: one size doesn't fit all, Nat. Rev. Molec. Cell Biol. 8: 284-95 (2007)].
Lysine acetylation is both regulated by, and regulates other, post-translational modifications, and is involved in the regulation of many cytoplasmic processes and nearly all nuclear functions. [Id; citing Choudhary, C. et al, Lysine acetylation targets protein complexes and co-regulates major cellular functions, Science 325: 834-40 (2009).] Post translational modifications may prevent or lead to a subsequent modification on histone and non-histone proteins by either recruitment or obstruction of binding proteins. [Id, citing Latham, J A, and Dent, S Y, Cross-regulation of histone modifications, Nat. Struct. Molec. Biol. 14: 1017-24 (2007); Yang, X J and Seto E, Lysine Acetylation codified cross talk with other posttranslational modifications, Molec. Cell 31: 449-61 (2008).]
HDACs
Four classes of mammalian histone deacetylases (HDACs) have been identified based on sequence similarity to yeast counterparts. [Id., citing de Ruijter, A J et al, Histone deacetylases (HDACs): characterization of the classical HDAC family, Biochem. J. 370: 737-49 (2003); Gregoretti, I V et al, Molecular evolution of the histone deacetylase family: functional implications of phylogenetic analysis, J. Mol. Biol. 338: 17-31 (2004)]. HDACs from classes I, II, and IV are dependent on Zn2+ for deacetylase activity. Class I HDACs include HDAC1, HDAC2, HDAC3 and HDAC8, and are ubiquitously expressed nuclear enzymes. [Id., citing de Ruijter, A J et al, Histone deacetylases (HDACs): characterization of the classical HDAC family, Biochem. J. 370: 737-49 (2003)]. Class I HDACs, with the exception of HDAC8, are components of a multiprotein complex.
Class I HDACs have been shown, via knock out studies, to be involved in cell proliferation and survival. [Id., citing Marks, P A, “Histone deacetylase inhibitors: a chemical genetics approach to understanding cellular functions<” Biochim. Biophys. Acta 1799: 717-25 (2010); Haberland, M. et al, “The many roles of histone deacetylases in development and physiology: implications for disease and therapy,” Nat. Rev. Genet. 10: 32-42 (2009)] HDAC1 and 2 are capable of forming homo- and hetero-dimers with each other, which is required for HDAC activity and which allows the HDACs to act together or separately from one another. [Id citing Taplick, J et al, “Homo-oligomerisation and nuclear localization of mouse histone deacetylase 1,” J Molec. Biol. 308: 27-38 (2001); Luo, Y et al, “Transregulation of histone deacetylase activities through acetylation,” J. Biol. Chem. 284: 34901-34910 (2009)] HDAC1 and 2 are each found in multiprotein corepressor complexes, including Sin3, nucleosome-remodeling HDAC (NuRD) and CoREST. Such complexes are recruited to regulatory regions of chromatin by various transcription factors, such as Sp1, Sp3, p53, NF-B and YY1, and have diverse and specific roles. [de Ruijter, A J et al, “Histone deacetylases (HDACs): characterization of the classical HDAC family,” Biochem. J. 370: 737-49 (2003); Yang, X J and Seto, E, “The Rpd3/Had 1 family of lysine deacetylases from bacteria and yeast to mice and men,” Nat. Rev. Molec. Cell Biol. 9: 206-218 (2008)] The Sin3 core complex comprises many proteins, including HDAC 1 and 2, and serves as a scaffold for the addition of other functional protein modules involved in nucleosome remodeling, DNA methylation, histone methylation and N-acetylglucosamine transferase activity. [Id citing Silverstein, R A and Ekwall, K, “Sin3: a flexible regulator of global gene expression and genome stability,” Curr. Genet. 47: 1-17 (2005); Hayakawa, T and Nakayama, J, “Physiological roles of class I HDAC complex and histone demethylase,” J. Biomed. Biotechnol. 2011: 129383 (2011)].
The NuRD complex comprises HDAC1 and HDAC2, which act as HDACs, and Mi-2a and/or Mi-2b, which provide(s) an ATP-dependent chromatin-remodeling function. Various other components of the NuRD complex include RbAp46/RbAp48, p66a or p66b, and methyl-CpG-binding domain-containing proteins (MBD2 or MBD3). Only MBD2 is able to recognize methylated DNA. [Id., citing Hayakawa, T and Nakayama, J, “Physiological roles of class I HDAC complex and histone demethylase,” J. Biomed. Biotechnol. 2011: 129383 (2011); Denslow, S A and Wade P A, “The human Mi-2/NuRD complex and gene regulation,” Oncogene 26: 5433-5438 (2007)] Also, identified as a component of NuRD is the lysine-specific demethylase 1, KDM1/LSD1. [Id., citing Wang, Y. et al, “LSD1 is a subunit of the NuRD complex and targets the metastasis program in breast cancer,” Cell 138: 660-672 (2009)].
HDAC1 and HDAC2 have also been identified as components of Nanog- and Oct4-associated deacetylase (NODE) complex. This complex also includes MTA1 or MTA2, p66a or p66b, but not the histone-binding proteins RbAp46/RbAp48 and the helicase-like ATPase Mi-2. The NODE complex has been identified as being a repressor of Nanog and Oct4 genes, thereby controlling embryonic stem cell differentiation. [Id citing Liang, J. et al, “Nanog and Oct4 associate with unique transcriptional repression complexes in embryonic stem cells,” Nature Cell Biol. 10: 731-39 (2008)].
The CoREST complex also includes HDAC1 and HDAC2, but comprises proteins different from those of Sin3 and NuRD. KDM1/LSD1, as a component of the CoREST complex, promotes demethylation of H3 dimethylated on lysine 4 (H3K4me2). This, in turn, results in formation of a repressive chromatin structure. [Lee, M G et al, “An essential role for CoREST in nucleosomal histone 3 lysine 4 demethylation,” Nature 437: 432-35 (2005); Shi, Y J et al, “Regulation of LSD1 Histone demethylase activity by its associated factors,” Molec. Cell. 19: 857-64 (2005)]. CoREST acts as a coactivator of transcription in embryonic and neural stem cells by recruiting H3K4 methyltransferase to the RE1 sites of target genes, but acts as corepressor in terminally differentiating nonneuronal cells by recruiting KDM1/LSD1 to demethylate H3K4me2 and the methyltransferase G9a to methylate H3K9 at the RE1 sites of target genes. [Id citing Cunliffe, V T, “Eloquent silence: developmental functions of class I histone deacetylases,” Curr. Op. Genetic Dev. 18: 404-410 (2008)]. CoREST can also associate with other proteins to form larger complexes, such as ZNF217 (a Krüppel-like zinc finger protein that is a proposed oncogene product in breast cancer) or with other complexes, such as C-terminal binding protein (CtBP) complex and chromatin-remodeling complex, SWI/SNF. [Id citing Hayakawa, T and Nakayama, J, “Physiological roles of class I HDAC complex and histone demethylase,” J. Biomed. Biotechnol. 2011: 129383 (2011); Battaglia, S. et al, “Transcription factor co-repressors in cancer biology: roles and targeting,” Intl J. Cancer 126: 2511-19 (2010)].
Class II HDACs are further grouped into subclass IIa (HDAC4, HDAC5, HDAC7 and HDAC9) and subclass IIb (HDAC6 and HDAC10). Class II HDACs are expressed in a tissue specific manner, and are shuttled between the cytoplasm and nucleus. [Id citing Marks, P. A., Histone deacetylase inhibitors: a chemical genetics approach to understanding cellular functions,” Biochim. Biophys. Acta 1799: 717-25 (2010); Haberland, M. et al, “The many roles of histone deacetylases in development and physiology: implications for disease and therapy,” Nat. Rev. Genet. 10: 32-42 (2009); Yang, X J and Seto E., “The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men,” Nat. Rev. Molec. Cell Biol. 9: 206-18 (2008); Verdin, E et al, “Class II histone deacetylases: versatile regulators,” Trends Genet. 19: 286-93 (2003)].
Subclass IIa HDACs acts as signal transducers characterized by conserved serine residues in the N-terminal regulatory domain that is reversibly phosphorylated. Phosphorylation of those residues results in an interaction with the 14-3-3 proteins, and results in the export of HDACs and the expression of target genes. Several different kinases and phosphatases have been shown to regulate the trafficking of class IIa HDACs from the cytoplasm to the nucleus. [Id citing Parra, M and Verdin E, “Regulatory signal transduction pathways for class IIa histone deacetylases,” Curr. Opin. Pharmacol. 10: 454-60 (2010)]. Some investigators have proposed that class IIa HDACs may act as bromodomains under some circumstances, and thereby recognize lysine in a sequence dependent manner and regulate transcription by recruiting chromatin modifying enzymes. [Id, citing Bradner, J E et al, “Chemical Phylogenetics of histone deacetylases,” Nat. Chem. Biol. 6: 238-43 (2010)]. Class IIa HDAC may interact with MEF2 to provide additional targeting for the SMRT-NCoR complex, and may interact with numerous other transcription factors. [Id citing Fischle, W. et al, “Enzymatic activity associated with class II HDACs is dependent on a multiprotein complex containing HDAC3 and SMRT/N-CoR,” Molec. Cell 9: 45-57 (2002); Martin M et al, “Class IIa histone deacetylases: regulating the regulators,” Oncogene 26: 5450-67 (2007); Martin M. et al, “Class IIa histone deacetylases: conducting development and differentiation,” Int. J. Dev. Biol. 53: 291-301 (2009)].
Class IIb HDACs (HDAC6 and HDAC10) have fully or partially duplicated catalytic domains. Class IIb HDACs are primarily localized to the cytoplasm, but may shuttle to the nucleus as well. HDAC6 has been identified as involved in microtubule and actin-dependent cell motility via a tubulin deacetylase and a cortactin deacetylase function. HDAC6 also functions to clear misfolded protein through formation of aggresomes or autophagy [Id citing Yang, X J and Seto E, “Lysine Acetylation codified cross talk with other posttranslational modifications,” Molec. Cell 31: 449-61 (2008)].
HDACs are found to be upregulated or inappropriately recruited to DNA in several types of cancer. [Id, citing Witt, O et al, “HDAC family: what are the cancer relevant targets?”, Cancer Lett 277: 8-21 (2009); Marks, P. A., Histone deacetylase inhibitors: a chemical genetics approach to understanding cellular functions,” Biochim. Biophys. Acta 1799: 717-25 (2010)].
Transcriptional Reprogramming by Histone Deacetylase Inhibitors
Several HDAC inhibitors exist for treatment of cancers, cardiovascular diseases, neurodegenerative disorders and pulmonary diseases, and it is believed that the therapeutic benefit is derived from transcription reprogramming. [Haberland, M. et al, “The many roles of histone deacetylases in development and physiology: implications for disease and therapy,” Nat. Rev. Genet. 10: 32-42 (2009)]. Approximately 5% to 20% of genes are affected by the inhibition of HDAC activity, with equal numbers being up- and down-regulated. [Id citing Smith, K T and Workman, J L; “Histone deacetylase inhibitors: anticancer compounds,” Intl J. Biochem. Cell Biol. 41: 21-25 (2009)]. Most of those effects are the result of downstream consequences of direct transcriptional regulation, such as inhibition of transcription factor deacetylation causing changes to transcription factor-DNA binding. [Id citing Glozak, M A, et al, “Acetylation and deacetylation of non-histone proteins,” Gene 363: 15-23 (2005)]. Recent reviews have addressed biological function and gene expression changes as a result of HDAC inhibition. [Marks, P. A., Histone deacetylase inhibitors: a chemical genetics approach to understanding cellular functions,” Biochim. Biophys. Acta 1799: 717-25 (2010), Waanczyk, et al, “HDACi: going through the mechanisms,” Front. Biosci. 16: 340-59 (2011)].
Cancer
Cancer is a disease in which cells multiply without control and can invade nearby tissue. There are several main types of cancer categorized by the originating tissue of the invasive cells. For example, cancers arising from epithelial cells are termed “carcinomas”; cancers arising from connective tissue or muscle are termed “sarcomas”; and cancers arising from hemopoetic cells are termed “leukemias”. Each broad category of cancer comprises many different subtypes according to specific cell type, location in the body, and structural morphology. [Alberts et al., Molecular Biology of the Cell, 4th Ed. 2002]
A neoplasm is an abnormal mass of tissue that is caused by cells dividing more often than they should or that do not die as often as they should. These masses of cells may be benign (not cancer) if they reproduce without normal restraints on cell division, but do not invade surrounding tissue. The neoplasm may be malignant (cancer) if the cells can invade and colonize tissues normally reserved for other cell types. The invasiveness of malignant cells often involves individual cells migrating away from the tumor mass, entering the blood stream or lymph vessels, and migrating to another location to form secondary, metastatic tumor masses. [Alberts et al., Molecular Biology of the Cell, 4th Ed. 2002]
A growing body of scientific literature shows that epigenetic mechanisms such as DNA methylation, histone modification, nucleosome positioning, and micro-RNA expression are involved in the mechanism(s) underlying some types of cancer. Studies have shown that hypermethylation of tumor suppressor genes is associated with deacetylation of histones H3 and H4, loss of H3K4 trimethylation, and an increase in H3K9 methylation and H3K27 trimethylation. [Maria Berdasco, Aberrant Epigenetic Landscape in Cancer: How Cellular Identity Goes Awry, Developmental Cell, Volume 19, Issue 5, 16 Nov. 2010, Pages 698-711 (citing P. A. Jones, S. B. Baylin, The epigenomics of cancer, Cell, 128 (2007), pp. 683-692)] Other studies have shown that a loss of histone H4 mono- and tri-methylation lysine is associated with human and mouse tumors. [Id. citing M. F. Fraga, et al., Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer, Nat. Genet., 37 (2005), pp. 391-400.] Yet further studies have shown that two disease subtypes of prostate cancer, each with distinct risk of tumor recurrence, is associated with the acetylation and demethylation of five different residues of histones H3 and H4. [Id. citing M. F. Fraga, et al., Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer, Nat. Genet., 37 (2005), pp. 391-400.]
Several aberrant histone modifier genes that are tissue specific and which have been associated with cancer have been reported. For example, aberrant genes have been found in the classes of histone acetyl transferases (HATs), histone methyltransferases (HMTs), histone deacetylases (HDACs), and histone demethylases (HDMTs). The aberrations of those histone modifier genes include amplifications, mutations, translocations, hypermethylations, over-expressions, and deletions. [Id. citing M. F. Fraga, et al., Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer, Nat. Genet., 37 (2005), pp. 391-400.]
The link between cancer and DNA methylation has been known since at least 1983, when it was discovered that the genomes of cancer cells are hypomethylated relative to their normal counterpart tissue. In addition to genome wide hypomethylation, cancer cells may display site-specific CpG island promoter hypermethylation, thereby inactivating transcription. Many of the main biochemical pathways are disrupted by hypermethylation, such as “DNA repair [hMLH1 (mismatch repair gene 1), MGMT (O6-methylguanine-DNA methyltransferase), WRN (Werner syndrome, RecQ helikase like), BRCA1 (breast cancer 1)], cell cycle control (p16 INK4a, p15 I NK4b, RB), Ras signaling [RASSF1A [Ras association (RalGDS/AF-6) domain family member 1], NOREIA], apoptosis [TMS1 (target of methylation-induced silencing 1), DAPK1 (death-associated protein kinase), WIF-1, SFRP1], metastasis [cadherin 1 (CDH1), CDH13, PCDH10], detoxification [GSTP1 (glutathione S-transferase pi 1)], hormone response (ESR1, ESR2), vitamin response [RARB2 (retinoic acid receptor b2), CRBP1] and p53 network [p14 ARF, p73 (also known as TP73), HIC-1] among others.” [R Kanwala and S Gupta, Epigenetic modifications in cancer, Clin Genet. 2012 April; 81(4): 303-311.] Hypomethylation of tumor cells results in genome instability and disruption of imprinting patterns. Hypomethylation of cancer cells is commonly associated with oncogenes, such as c-Myc, and can potentially result in oncogene activation. A common loss of imprinting, which results from hypomethylation, is to insulin-like growth factor 2 (IGF2), which has been found in breast, liver, lung, and colon cancer. [Id.]
It has also been reported that human tumors have altered patterns of histone modification, and that such modifications are predictive of gene expression patterns. For example, the local gene expression of human tumors with increased levels of promoter region H3K4me3, H3K27ac, H2BK5ac and H4K20me1 and increased gene body region H3K79me1 and H4K20me1 is associated with high rates of transcription. [Id. citing Karlić R, Chung H R, Lasserre J, Vlahovicek K, Vingron M, Histone modification levels are predictive for gene expression, Proc Natl Acad Sci USA. 2010 Feb. 16; 107(7):2926-31.] Furthermore, HDAC mutation and over expression has been found to be associated with loss of acetylation on tumors. The inappropriate expression of histone methyl transferases and histone demethylases has been found associated with various types of cancer. [Id. citing Chi P, Allis C D, Wang G G, Covalent histone modifications—miswritten, misinterpreted and mis-erased in human cancers, Nat Rev Cancer. 2010 July; 10(7):457-69.] One study showed that mutations that inactivate the histone methyltransferase SETD2, the histone demethylase UTX, and JARID1C are found in renal carcinoma. [Id. citing Dalgliesh G L, et al., Systematic sequencing of renal carcinoma reveals inactivation of histone modifying genes, Nature. 2010 Jan. 21; 463(7279):360-3.] The trimethylation of H3K4 is predictive of prostate-specific antigen serum level elevation after prostatectomy for cancer, and H3 acetylation and H3K9 dimethylation is indicative of cancerous and nonmalignant prostate tissue. [Id. citing Lopez-Serra P, Esteller M. DNA methylation-associated silencing of tumor-suppressor microRNAs in cancer. Oncogene. 2011; 16:1-14.] The expression of EZH2 (enhancer of zeste homolog 2) has been found to be a prognostic indicator of aggressiveness of prostate, breast, and endometrial cancers. [Id. citing Bachmann I M, et al., EZH2 expression is associated with high proliferation rate and aggressive tumor subgroups in cutaneous melanoma and cancers of the endometrium, prostate, and breast, J Clin Oncol. 2006 Jan. 10; 24(2):268-73.]
Several studies have noted an effect of histone deacetylation and methylation inhibitors, such as trichostatin A (TSA), SAHA, DZNep, and BIX-01294, on tumors. [Wisnieski F, Calcagno D Q, Leal M F, Chen E S, Gigek C O, Santos L C, et al. Differential expression of histone deacetylase and acetyltransferase genes in gastric cancer and their modulation by trichostatin A. Tumour Biol 2014; 35:6373-81; Murphy S P, Lee R J, McClean M E, Pemberton H E, Uo T, Morrison R S, et al. MS-275, a class I histone deacetylase inhibitor, protects the p53-deficient mouse against ischemic injury. J Neurochem 2014; 129:509-15; Girard N, Bazille C, Lhuissier E, Benateau H, Llombart-Bosch A, Boumediene K, et al. 3-Deazaneplanocin A (DZNep), an inhibitor of the histone methyltransferase EZH2, induces apoptosis and reduces cell migration in chondrosarcoma cells. PLoS One 2014; 9:e98176; Savickiene J, Treigyte G, Stirblyte I, Valiuliene G, Navakauskiene R. Euchromatic histone methyltransferase 2 inhibitor, BIX-01294, sensitizes human promyelocytic leukemia HL-60 and NB4 cells to growth inhibition and differentiation. Leuk Res 2014; 38:822-9; Cortez C C, Jones P A. Chromatin, cancer and drug therapies. Mutat Res 2008; 647:44-51.
Electrochemical Properties of Living Tissue
A living tissue functions as an electrical machine, and the structure of cells comprising the tissue exhibit electrical properties including, but not limited to, the ability to conduct electricity, create electric fields, and function as electrical generators. The primary charge carriers in living organisms are negatively charged electrons, positively charged hydrogen protons, positively charged sodium, potassium, calcium and magnesium ions and negatively charged anions, particularly phosphate ions. (Reviewed in Haltiwanger, S., “The Electrical Properties of Cancer Cells,” July 2010, accessed from royalrife.com/haltiwanger1).
The body uses the exterior cell membrane, and positively charged mineral ions that are maintained in different concentrations on each side of the cell membrane, to create a cell membrane potential (a voltage difference across the membrane) and a strong electrical field around the cell membrane. (As used herein the term “electrical field” refers to the effect which a charged particle or body exerts on charged particles or bodies situated in the medium surrounding it; i.e., if a negatively charged particle is placed within the electric field of a positively charged particle, there will be an attractive force, while there will be a repulsive force if the charges are alike. The electric field is perpendicular to the magnetic field). This electrical field is a readily available source of energy for cellular activities, such as membrane transport, and the generation of electrical impulses in the brain, nerves, heart and muscles. The storage of electrical charge in the membrane and the generation of an electrical field create a battery function so that the liquid crystal (meaning symmetrically packed) electroactive intermediates and catalysts can transfer membrane charge to DNA. The body also uses the mitochondrial membrane and positively charged hydrogen ions to create a strong membrane potential across the mitochondrial membrane. Hydrogen ions are maintained in a high concentration on the outside of the mitochondrial membrane by the action of the electron transport chain (Haltiwanger, S., “The Electrical Properties of Cancer Cells,” July 2010, accessed from royalrife.com/haltiwanger1).
Animal cells are organized structures with an internal architecture of cytoskeletal proteins that connects all components of the cell. Cellular components do not randomly float around in the cell but are attached to the cytoskeletal framework and the membranes. Cytoskeletal filaments and tubules form a continuous system that links the cell surface to all organelle structures including passage through the nuclear membrane to the chromosomes. The liquid crystal proteins that compose the cytoskeleton support, stabilize and connect the liquid crystal components of the cell membrane with other cell organelles. The cytoskeletal proteins have multiple roles. They serve as mechanical scaffolds that organize enzymes and water and anchor the cell to structures in the extracellular matrix (ECM) via linkages through the cell membrane, and are dynamic network structures that create a fully integrated electronic that structurally and electronically links and integrates the proteins of the extracellular matrix with the cell organelles. (Haltiwanger, S., “The Electrical Properties of Cancer Cells,” July 2010, accessed from the royalrife.com/haltiwanger1).
The cytoskeleton is also attached through cell membrane connectors to liquid crystal protein polymers located in the external extracellular matrix (ECM) and to other cells. The liquid crystal protein polymers of the ECM are mostly composed of collagen, elastin, hylauronic acid, and interweaving glycoproteins such as fibronectin. The ECM is a transit area for the passage of nutrients from the bloodstream into the cells, for toxins released by the cells that pass through to the bloodstream, and for migrating immune cells involved in inflammatory reactions that secrete cytokines and other inflammatory mediators. (Haltiwanger, S., “The Electrical Properties of Cancer Cells,” July 2010, accessed from the royalrife.com/haltiwanger1).
Biochemically, the ECM is a metabolically and electrically active space that is involved in regulating cell growth control. Cellular components of the ECM are involved in the local production of growth factors, growth inhibitors and cytokines that affect the growth and metabolic activity of tissue/organ cells. (Reichart, L. F., “Extracellular matrix molecules,” In “Guidebook to the Extracellular Matrix, Anchor, and Adhesion Proteins,” (ed. T. Kreis and R. Vale). Oxford, England: Oxford University Press, pgs. 335-344, 1999).
Cells are electromagnetic in nature, and are capable of generating their own electromagnetic fields and of harnessing external electromagnetic energy of the right wavelength to communicate, control and drive metabolic reactions. Communications in living organisms are accomplished by chemical communication through the circulatory system and energetic communication through the nervous system. A solid state electronic communication system has also been hypothesized to operate in series and in parallel with the nervous system through the liquid crystal protein polymer connective system continuum of the cytoskeleton and extracellular matrix. It has been hypothesized that this continuum of liquid crystal connections function as electronic semiconductors and fiberoptic cables allowing the shunting of charge and associated electronic energy in and out of the cell. (Haltiwanger, S., “The Electrical Properties of Cancer Cells,” July 2010, accessed from the royalrife.com/haltiwanger1).
Most molecules in the body are electrical dipoles capable of oscillations and resonance. Electric fields induce or cause alignment of dipole moments. A dipole moment is a function of polarization processes and the strength of the electric field. When biological tissue is exposed to an electric field in the right frequency and amplitude windows, a preferential alignment of dipoles becomes established. Since the cell membrane contains many dipole molecules, an applied electric field causes a preferential alignment of the dipoles. Both internally generated and externally applied electromagnetic fields can affect cell functions. (Haltiwanger, S., “The Electrical Properties of Cancer Cells,” July 2010, accessed from the royalrife.com/haltiwanger1).
Electrical Properties of the Cell in Disease Repair and Healing
The body uses electricity (biocurrents) as part of its mechanism for controlling growth and repair. Some of these biocurrents travel through hydrated liquid crystal semiconducting (the term “semiconductor” refers a material whose conductivity lies between that of an electrical conductor, such as a metal, and an insulator) protein-proteoglycan (collagen-hyaluronic acid) complexes of the ECM. It has been hypothesized that biocurrents in the ECM pass through the cell membrane into the cell and electrons produced in the cell also pass out through the cell membrane. The biological liquid crystal molecules and structures such as hyaluronic acid, prothrombin, DNA, cytoskeletal proteins and cell membranes maintain both an inward and outward current. The inward current flows from the cell membrane to cell structures like mitochondria and DNA, and the outward current flows back along liquid crystal semiconducting cytoskeletal proteins through the cell membrane to the ECM. Electrical charges stored in the cell membrane (capacitance) and electrical charges of oxygen free radicals are normally transferred to DNA and are involved in DNA activation and the creation of an electrical field around DNA. DNA is very effective in transferring large amounts of electrical charge along its long axis. (Haltiwanger (2010) citing Garnett M., “First Pulse: A Personal Journey in Cancer Research,” New York, N.Y.: First Pulse Projects, 1998). An alternating current oscillating circuit between the cell membrane and DNA conducted over the electronic liquid crystal network of the cell is thought to be involved in cellular processes such as gene expression. (Garnett, M. and Remo, J. L., “DNA Reductase: A Synthetic Enzyme with Opportunistic Clinical Activity Against Radiation Sickness,” International Symposium on Applications of Enzymes in Chemical and Biological Defense, Orlando, Fla., May, 2001, p. 41.)
It has also been hypothesized that electrical pathways between the cell membrane and DNA are related to cell development, and use anaerobic mechanisms of ATP production. This natural electrical pathway is thought to be transiently disrupted in healthy cells that are involved in wound healing and inflammation, and permanently disrupted in cancer cells that rely on anerobic glycolysis for energy production. (Haltiwanger (2010) citing Garnett M., “First Pulse: A Personal Journey in Cancer Research,” New York, N.Y.: First Pulse Projects, 1998).
One feature that is characteristic of cancer cells is that they have a lower membrane potential than those of healthy adult cells. In addition to cancerous cells having a lower electrical potential, the electrical connections of cancer cells are disrupted. (Haltiwanger (2010) citing Cone, 1975) This may result in several phenotypic traits of cancer cells; i.e. cancer cells are more easily detached, do not exhibit contact inhibition growth patterns, and have signaling and growth mechanisms independent of normal tissue.
Glycoproteins secreted from the cell interior and cellular components of the ECM produce a glycocalyx that covers the cells. These glycoproteins characteristically have a negative charge. The negative charges of the ECM-glycocalyx interface help determine water balance, ion balance and osmotic balance both in the ground substance of the ECM and inside the cells. ECM proteoglycans exist in fern shapes that allow electric charges to flow, and in disorganized shapes that impair transit through the ECM of electrical currents and nutrients. These disorganized shapes occur in the presence of tissue inflammation and toxins, such as free radicals, reactive oxygen species (e.g. superoxide, peroxide, or hydroxyl ions) in the ECM. Such structures produce pockets of high electrical resistance. (Haltiwanger (2010)).
Some researchers have proposed that the electrical charges stored across the cell lipid bilayer and oxygen free radicals are potentially transferred to molecules of DNA. The transfer of charge along DNA, which is very capable of transferring charge along its axis, may result in activation of DNA. It is possible that the electrical pathway from the cell's lipid bilayer to DNA is related to normal cell functions, such as development and aerobic ATP production. Thus, when the natural electrical pathway of charge is permanently disrupted, it disrupts those normal cell functions.
Measuring Electrical Properties of Biological Tissue
The electrical properties of biological tissue can be measured when current flows through the tissue by a phenomenon termed “impedance” or alternatively “bioimpedance”, which refers to an opposition to the flow of alternating current through a conductor, and is described by a relation between voltage and current in a system. (Holder, D. S., “Appendix A: A brief introduction to bioimpedance,” in “Electrical Impedance Tomography”, Institute of Physics Publishing, Bristol and Philadelphia (2005), pp. 411-422). Impedance is defined as the ratio of incremental change in voltage to the resulting current (or vice versa) across an electrochemical cell or an electrical circuit. (Crescentini, M. et al., “Recent trends for (bio)chemical impedance sensor electronic interfaces,” Electroanalysis, 24(3): 563-572 (2012)).
Impedance can be measured in tissues and cells using electrochemical impedance spectroscopy (EIS). Through the application of a small sinusoidally varying potential U, one measures the resulting current response I. By repeating the process at varying excitation frequencies f, impedance can be calculated as a function of the angular frequency ω, given by the relationship:
            Z      ⁡              (                  j          ⁢                                          ⁢          ω                )              =                            U          ⁡                      (                          j              ⁢                                                          ⁢              ω                        )                                    I          ⁡                      (                          j              ⁢                                                          ⁢              ω                        )                              =                                    Z            r                    ⁡                      (            ω            )                          +                              jZ            i                    ⁡                      (            ω            )                                ,where ω=2Πf. (Grieshaber, D. et al., “Electrochemical biosensors,” Sensors, 8: 1400-1458 (2008)).
More specifically, when applying a sinusoidal voltage reference Vref(t)=|Vref|sin (ω0t) across the cell and assuming linear behavior, the corresponding current flowing through the cell is I(t)=|I|sin (ω0t+θ), wherein θ is the phase shift of the signal with respect to the excitation. Thus, the relationship between excitation and readout signals depends only on phases and amplitude ratios. (Crescentini, M. et al., “Recent trends for (bio)chemical impedance sensor electronic interfaces,” Electroanalysis, 24(3): 563-572 (2012)). Therefore, impedance is made of two components: resistance or the real part of the data, and reactance, the out-of-phase data. (Crescentini, M. et al., “Recent trends for (bio)chemical impedance sensor electronic interfaces,” Electroanalysis, 24(3): 563-572 (2012)).
Resistance (R) is a measure of the extent to which a substance opposes the flow of electrons or, in aqueous solution as in living tissue, the flow of ions among its cells. The three fundamental properties governing the flow of electricity are “voltage”, “current” and “resistance”. Voltage is the pressure exerted on a stream of charged particles moving down a wire or through an ionized salt solution. Current is the amount of charge flowing per unit time. Resistance is the ease or difficulty with which the charged particles can flow. Voltage, current and resistance are related by Ohm's law: V (voltage, Volts)=I (current, Amps)×R (resistance, Ohms (Ω)). Ohm's law applies to both direct current (d.c. or steadily flowing) or alternating current (a.c. or current that flows backwards and forwards).
Capacitance (C) refers to the extent to which an electronic component, circuit, or system stores and releases energy as the current and voltage fluctuate with each AC cycle. The capacitance physically corresponds to the ability of plates in a capacitor to store charge. With each cycle, charges accumulate and then discharge. Direct current cannot pass through a capacitor. Alternating current can pass because of the rapidly reversing flux of charge. The capacitance is an unvarying property of a capacitive or more complex circuit. However, the effect in terms of the ease of current passage depends on the frequency of the applied current; charges pass backwards and forwards more rapidly if the applied frequency is higher.
Reactance (X), analogous to resistance, refers to the current travelling through a capacitor or a coil. A higher reactance has a higher effective resistance to alternating current. Like resistance, its value is in Ohms, but it depends on the applied frequency, and is described by the relation: Reactance (Ohms)=1/(2×r×Frequency (Hz)×Capacitance (Farads)). When a current is passing through a purely resistive circuit, the voltage recorded across the resistor will coincide exactly with the timing, or phase, of the applied alternating current. However, when current flows across a capacitor, the voltage recorded across it lags behind the applied current because of back and forth flow of current requiring alternating charging and discharging of the plates of the capacitor. In terms of a sine wave which has 360° in a full cycle, the lag is one quarter of a cycle, i.e., 90°.
Impedance is the frequency dependent resistance derived from the following three components of an AC circuit: direct current (DC) resistance; capacitive resistance; and inductive reactance. Capacitance is produced by storing charge on a surface at an energy expense producing a retardation of voltage flow. Inductance is produced by storing energy in a magnetic field in bulk space at an energy expense producing a retardation of current flow. Capacitance is counted in Farad units and inductance in Henry units. These two retardation effects are combined in a process and representation called the phase angle, which is the angular summation of the two waves or pulses of voltage and current. In the Mott-Schottky form of impedance measurement, only a single frequency influence is used. This is a departure from Nyquist or Cole (Cole, K. S. and Cole, R. H, “Dispersion and Absorption in Dielectrics. I. Alternating Current Characteristics,” J. Chem. Phys. 9: 341-351 (1941)) plots, which utilize a descending frequency series. The Mott-Schottky method is useful for analysis of the underlying impedance vectors within devices and within molecules.
Vortex Theory
Vortex theory is widely used in physics (Ginzburg-Landau theory, superconductivity, spiral galaxies, black holes, aerodynamics, hydrodynamics), but appears to be rarely used in molecular biology. The superconducting vortex lattice was first predicted by the Nobel laureate Alexei Abrikosov, who predicted that a vortex supercurrent circulates around the core of a magnetic vortex due to the circulating supercurrent's production of magnetic fields. [Abrikosov, A. A. On the magnetic properties of superconductors of the second group. Soviet Phys. JETP 5, 1174-1182 (1957); Abrikosov, A. A., The magnetic properties of superconducting alloys, Journal of Physics and Chemistry of Solids, 2(3), 199-208, 1957]. Typically, the arrangement of the small spiraling magnetic vortices is an orderly honeycomb lattice array.
Vortex theory facilitates understanding the equilibrium geometry between magnetic spirals and their unexpected precursors, hexagonal lattices. These forms may be produced in various mediums such as Aluminum-Gallium-Arsenic (Al—Ga—As) layers by underlying vector forces as described in descriptions of vortex production in phase-dependent circle forms and hexagonal honeycomb lattice forms. [Tosi, G. et al. Geometrically locked vortex lattices in semiconductor quantum fluids. Nat. Commun. 3:1243 doi: 10,1038/ncomms2255 (2012).] In such a medium, pulsed laser mirror array excitations of highly defined layers of Al—Ga—As in semiconductor microcavities produces excited state photons called polaritons. These quasi-particle pump fields sculpture the resulting condensates. The condensate forms, the selection of which was controlled by blue-shifting the excitations, are those of both hexagonal honeycomb lattices and vortex and anti-vortex circles. These optical systems are equivalent to one and two dimensional spin systems. [Id.]
Physical-chemistry studies in iridium complexes showed the association between the hexagonal honeycomb lattice and the spiral magnetic field [Kimchi, I., Coldea, R., Vishwanath, A., Unified theory of spiral magnetism in the harmonic-honeycomb iridates, alpha, beta, gamma, Li2IrO3, arXiv: 1408.3640v3 (cond-matter.str-el) 15 Jun. 2015; K. A. Modic et al., Realization of a three-dimensional spin-anisotropic harmonic honeycomb iridate, Nature Communications 5, Art. no. 4203, doi:10,1038/ncomms5203, June 2014.]
Other studies investigating the organization transitions of vortices under the influence of magnetic field, current, and temperature, have shown that many interactive vortex structures may be produced, including clusters, chains, and mazes. Tunable effects produce transitions from short-range clustering to long range order such as parallel chains, gels, glasses, and crystalline lattices. [L. Komendova, M. V. Milosevic, and F. M. Peeters, Soft vortex matter In a type-I/type-II superconducting bilayer, Phys. Rev. B 88, 094515 (2013)].
Ruthenium Compounds and Complexes
Ruthenium is a transition metal of group 8 of the periodic table. Its electronic configuration is 1s2 2s2p6 3s2p6d10 4s2p6d7 5s1. Ruthenium complexes have been described as having the capability to interact with DNA. [Hui Chao, DNA Interactions with Ruthenium(II) Polypyridine Complexes Containing Asymmetric Ligands, Bioinorg Chem Appl. 2005; 3(1-2): 15-28.] Certain ruthenium compounds have been described as having wide ranging biological effects on cells, and have been investigated for the development of disease therapies.
Ruthenium is capable of the widest range of oxidation states of any element, and Ru(II) and Ru(III) oxidation states are capable of six-coordinated octahedral configurations. Additional ligands are able to fine tune the steric and electronic properties of ruthenium complexes. [Ileana Dragutan, et al., Editorial of Special Issue Ruthenium Complex: The Expanding Chemistry of the Ruthenium Complexes, Molecules 2015, 20(9), 17244-17274.] The weak strength of particular metal-ligand bonds and the thermodynamic stability of Ru(III) complexes vs. Ru(II) complexes are also important and can effect ligand exchange kinetics. [Id. citing Strasser, S.; Pump, E.; Fischer, R. C.; Slugovc, C. On the chloride lability in electron-rich second-generation ruthenium benzylidene complexes. Monatsh. Chem. 2015, 146, 1143-1151.] By varying ligands ancillary to ruthenium complexes, it is possible to modulate their reduction/oxidation properties, which allows a large platform of ruthenium complexes having either achiral or chiral configurations. [Id. citing Gunanathan, C.; Milstein, D. Bond activation and catalysis by ruthenium pincer complexes. Chem. Rev. 2014, 114, 12024-12087; Tonnemann, J.; Scopelliti, R.; Severin, K. (Arene)ruthenium complexes with imidazolin-2-imine and imidazolidin-2-imine ligands. Eur. J. Inorg. Chem. 2014, 2014, 4287-4293; Ablialimov, O.; Kedziorek, M.; Maliñska, M.; Woźniak, K.; Grela, K. Synthesis, structure, and catalytic activity of new ruthenium(II) indenylidene complexes bearing unsymmetrical N-heterocyclic carbenes. Organometallics 2014, 33, 2160-2171; Mukherjee, T.; Ganzmann, C.; Bhuvanesh, N.; Gladysz, J. A. Syntheses of enantiopure bifunctional 2-guanidinobenzimidazole cyclopentadienyl ruthenium complexes: Highly enantioselective organometallic hydrogen bond donor catalysts for carbon-carbon bond forming reactions. Organometallics 2014, 33, 6723-6737; Dragutan, I.; Dragutan, V.; Verpoort, F. Carbenoid transfer in competing reactions catalyzed by ruthenium complexes. Appl. Organomet. Chem. 2014, 28, 211-215; Biffis, A.; Baron, M.; Tubaro, C. Poly-NHC Complexes of transition metals: Recent applications and new trends. Adv. Organomet. Chem. 2015, 63, 203-288; Ivry, E.; Ben-Asuly, A.; Goldberg, I.; Lemcoff, N. G. Amino acids as chiral anionic ligands for ruthenium based asymmetric olefin metathesis. Chem. Commun. 2015, 51, 3870-3873; Carreira, E. M.; Yamamoto, H. Synthetic Methods III—Catalytic Methods: C—C Bond Formation. In Comprehensive Chirality; Elsevier: Amsterdam, The Netherlands, 2012; Volume 4.]
Several ruthenium based metallodrugs, which rely on ruthenium chemistry, are able to differentially target tumor cells among healthy cells. Such compounds comprise dendrimers, dendronized polymers, protein conjugates, intelligent nanoparticles, and polymer Ru-complex conjugates. [Id. citing Valente, A.; Garcia, M. H. Syntheses of macromolecular ruthenium compounds: A new approach for the search of anticancer drugs. Inorganics 2014, 2, 96-114.] These ruthenium complexes are believed to function through mechanisms different from platinum drugs that are active through interaction with DNA. [Id. citing Spreckelmeyer, S.; Orvig, C.; Casini, A. Cellular transport mechanisms of cytotoxic metallodrugs: An overview beyond cisplatin. Molecules 2014, 19, 15584-15610; Adhireksan, Z.; Davey, G. E.; Campomanes, P.; Groessl, M.; Clavel, C. M.; Yu, H.; Nazarov, A. A.; Yeo, C. H. F.; Ang, W. H.; Droge, P.; et al. Ligand substitutions between ruthenium-cymene compounds can control protein versus DNA targeting and anticancer activity. Nat. Commun. 2014, 5, 3462; Nazarov, A. A.; Gardini, D.; Baquié, M.; Juillerat-Jeanneret, L.; Serkova, T. P.; Shevtsova, E. P.; Scopelliti, R.; Dyson, P. J. Organometallic anticancer agents that interfere with cellular energy processes: A subtle approach to inducing cancer cell death. Dalton Trans. 2013, 42, 2347-2350; Nazarov, A. A.; Meier, S. M.; Zava, O.; Nosova, Y. N.; Milaeva, E. R.; Hartinger, C. G.; Dyson, P. J. Protein ruthenation and DNA alkylation: Chlorambucil-functionalized RAPTA complexes and their anticancer activity. Dalton Trans. 2015, 44, 3614-3623; Sharma, A. R.; Gangrade, D. M.; Bakshi, S. D.; John, J. S. Ruthenium complexes: Potential candidate for anti-tumour activity. Int. J. Chem. Tech. Res. 2014, 6, 828-837; Guidi, F.; Modesti, A.; Landini, I.; Nobili, S.; Mini, E.; Bini, L.; Puglia, M.; Casini, A.; Dyson, P. J.; Gabbiani, C.; et al. The molecular mechanisms of antimetastatic ruthenium compounds explored through DIGE proteomics. J. Inorg. Biochem. 2013, 118, 94-99; Wu, K.; Luo, Q.; Hu, W.; Li, X.; Wang, F.; Xiong, S.; Sadler, P. J. Mechanism of interstrand migration of organoruthenium anticancer complexes within a DNA duplex. Metallomics 2012, 4, 139-148.] Other anti-tumor complexes of ruthenium are known. [Viktor Brabec, Olga Novakova, DNA Binding Mode of Ruthenium Complexes and Relationship to Tumor Cytotoxicity, ZP.tech online, www.mitochondrial.net/showabstract.php?pmid=16790363, June 2006; Jing Sun, Yongchao Huang, Chuping Zheng, Yanhui Zhou, Ying Liu, Jie Liu, Ruthenium (II) Complexes Interact with Human Serum Albumin and Induce Apoptosis of Tumor Cells, Biological Trace Element Research, V. 163, Issue 1-2, pp 266-274, February 2015; Jiao, W., et al., E2F-Dependent Repression of Topoisomerase II Regulates Heterochromatin Formation and Apoptosis in Cells with Melanoma-Prone Mutation, Cancer Res., 65:(10):4067-4077, May 2005.]
Ruthenium complexes are believed to act as redox-activatable prodrugs. [Id. citing Lippard, S. J.; Graf, N. Redox activation of metal-based prodrugs as a strategy for drug delivery. Adv. Drug Deliv. Rev. 2012, 64, 993-1004; Lee, H. Z. S.; Buriez, O.; Labbé, E.; Top, S.; Pigeon, P.; Jaouen, G.; Amatore, C.; Leong, W. K. Oxidative sequence of a ruthenocene-based anticancer drug candidate in a basic environment. Organometallics 2014, 33, 4940-4946.]
Ligands of biological origin are capable of minimizing toxicity of ruthenium compounds, enhancing their compatibility with a biological environment, and modulating coordination modes. Such biologically derived ligands include amino acids, peptides, proteins, carbohydrates, purine bases, oligonucleotides, and steroids, among others. [Id. citing Paul, L. E. H.; Furrer, J.; Therrien, B. Reactions of a cytotoxic hexanuclear arene ruthenium assembly with biological ligands. J. Organomet. Chem. 2013, 734, 45-52; Rathgeb, A.; Bohm, A.; Novak, M. S.; Gavriluta, A.; Domotor, O.; Tommasino, J. B.; Enyedy, E. A.; Shova, S.; Meier, S.; Jakupec, M. A.; et al. Ruthenium-nitrosyl complexes with glycine, 1-alanine, 1-valine, 1-proline, d-proline, 1-serine, 1-threonine, and 1-tyrosine: Synthesis, X-ray diffraction structures, spectroscopic and electrochemical properties, and antiproliferative activity. Inorg. Chem. 2014, 53, 2718-2729; Aman, F.; Hanif, M.; Siddiqui, W. A.; Ashraf, A.; Filak, L. K.; Reynisson, J.; Sohnel, T.; Jamieson, S. M. F.; Hartinger, C. G. Anticancer ruthenium(η6-p-cymene) complexes of nonsteroidal anti-inflammatory drug derivatives. Organometallics 2014, 33, 5546-5553; Kandioller, W.; Balsano, E.; Meier, S. M.; Jungwirth, U.; Goschl, S.; Roller, A.; Jakupec, A.; Berger, W.; Keppler, B. K.; Hartinger, C. G. Organometallic anticancer complexes of lapachol: Metal centre-dependent formation of reactive oxygen species and correlation with cytotoxicity. Chem. Commun. 2013, 49, 3348-3350; Pettinari, R.; Marchetti, F.; Condello, F.; Pettinari, C.; Lupidi, G.; Scopelliti, R.; Mukhopadhyay, S.; Riedel, T.; Dyson, P. J. Ruthenium(II)-arene RAPTA type complexes containing curcumin and bisdemethoxycurcumin display potent and selective anticancer activity. Organometallics 2014, 33, 3709-3715; Collins, I., Jones, A. M., Diversity-oriented synthetic strategies applied to cancer chemical biology and drug discovery. Molecules 2014, 19, 17221-17255.]
Complexes containing ruthenium and zinc have been reported, but none of these have been of pharmaceutical use. [Dornajafi, M, Characterization and Fabrication of Novel Ruthenium Oxide-Zinc Batteries, Electrical & Computer Engineering Theses and Dissertations, University of Maryland, 2010; Casimiro, D. R., Wong, L. L., Colon, J. L., Zewert, T. E., Richards, J. H., Chang, I. J., Winkler, J. R., Gray, H. B., Electron transfer in ruthenium/zinc porphyrin derivatives of recombinant human myoglobins. Analysis of tunneling pathways in myoglobin and cytochrome c, J. Am. Chem. Soc., 115 (4), pp 1485-1489, 1993.]
Electron Spin
According to quantum theory, each electron has a spin that is associated with an angular momentum leading to a magnetic moment. Consequently, the negative charge carried by the electron is also associated with a spin resulting in a circulating electric current. The circulating current induces a magnetic moment μS which, if the electron is subjected to a steady magnetic field H0, causes the electron to experience a torque that tends to align the magnetic moment with the field. The energy of the system depends upon the projection of the spin vector along H0. Quantum theory stipulates that only two values are permitted for an electron, which means that the electron magnetic moment can only assume two projections or spin states onto the applied field: the “+½ spin state”, when the electron's magnetic moment μS is aligned with the direction of the applied magnetic field H0; and the “+½ spin state”, when the electron's magnetic moment μS is aligned opposed to the direction of H0. Consequently, the ensemble of energy levels also reduces to two values, designated as E+, a lower energy level corresponding to the +½ spin state (aligned with the direction of the applied magnetic field) and E−, a higher energy level corresponding to the −½ spin state (opposed to the direction of the applied magnetic field). Because the +½ spin state is of slightly lower energy, in a large population of electrons, slightly more than half of the electrons will occupy this state, while slightly less than half will occupy the −½ spin state. The slight excess of the electron spin in the direction of the magnetic field constitutes a slight net magnetization of the material, a phenomenon known as spin polarization. The difference in energy between the two spin states increases with increasing strength of the magnetic field H0. The higher the strength of H0, the more the net magnetization or the spin polarization, i.e. the higher the number of electrons that will occupy the +½ state as compared to the −½ state.
In addition to the spinning motion, the angular momentum vector of a spinning electron as a result of the torque exhibits a precession around the external field axis with an angular frequency ωL. The precessional motion, known as Larmour precession, is similar to a spinning top whose spin axis rotates slowly around the vertical. The frequency of precession, ωL termed the Larmour frequency, is the number of times per second the electron precesses in a complete circle. The precessional frequency increases the strength of the magnetic field H0.
If an electron that is precessing in an applied magnetic field is exposed to electromagnetic radiation of a frequency ωA that matches with the precessional frequency ωL, the resulting condition is known as resonance. In the resonance condition, an electron of a lower energy +½ spin state (aligned with the applied magnetic field) will transition or flip to the higher energy −½ spin state (opposed to the applied magnetic field). In doing so, the electron absorbs radiation at this resonance frequency, ωA=ωL. This frequency corresponds to the separation between the energy levels of the two spin states, equal to ΔE=E+−E−. This phenomenon is called electron spin resonance (ESR). ESR measures a molecular splitting constant, which is the Gaussian distance or hyperfine shift between the repetitive peaks.
When stimulated by a reaction, the rate of precession can increase, and the dynamic effect is described as Rabi frequency. According to Maxwell-Faraday-Heaviside laws, a moving charge produces a magnetic field in its path, given by: Curl B=4 pi C, where Curl is the net circulating magnetic energy, and C is the charge density or rate of charge moving through a cross section of space or material.
When an atom or molecule has an even number of electrons, electron spins pair off in atomic or molecular orbitals so that virtually no net spin magnetism is exhibited; such material is said to be “diamagnetic”. However, when an atom or molecule has an odd number of electrons, complete pairing is not possible and the material is said to be “paramagnetic”. The phenomena of spin magnetism (spin polarization) and ESR are observed in paramagnetic materials. The minimally attracted spinning d-orbital electrons in ruthenium render paramagnetic properties in ruthenium complexes.
The motion of the rutheneum d-orbital electrons produces an intermittent or pulsed magnetic field. In suitable ruthenium complexes, the d-orbital electrons are capable of introducing long range molecular magnetic signals into chemical systems. Thus, ruthenium can form coordination complexes with suitable solubility, voltametric behavior, and oxidation state. The inorganic catalyst literature identifies the palladium-ruthenium system (Pd—Ru) (Tripathi, S. N., Bharadwaj, S. R., Dharwadkar, S. R., The Pd—Ru System (Palladium-Ruthenium), J. of Phase Equilibria, V. 14, No. 5, 638-642, 1993; Adams, R. D., Captain, B. F. W., Smith, M. D. Lewis Acid-Base Interactions Between Metal Atoms and their Applications for the Synthesis of Bimetallic Cluster Complexes, J. Am. Chem. Soc., V. 124, No. 20, 5628-9, May 2002) as having a singular peritectic phase with synergic effect on the catalytic hydrogenation of nitroaromatics (Wan, B. S., Liao, S. J., XU, Y., Yu, D. R., Synergic Effect of Palladium-Based Bimetallic Catalysts for the Hydrogenation of Nitroaromatics, Reaction Kinetics and Catalysis Letters, V. 63, No. 2, 397-401). In peritectic transformations, a liquid and solid of fixed proportions react to form a new microcrystal phase capable of nucleation and growth. Pd—Ru also has Lewis acid-base interactions between the metal atoms (Tripathi, S. N., Bharadwaj, S. R., Dharwadkar, S. R., The Pd—Ru System (Palladium-Ruthenium), J. of Phase Equilibria, V. 14, No. 5, 638-642, 1993; Adams, R. D., Captain, B. F. W., Smith, M. D. Lewis Acid-Base Interactions Between Metal Atoms and their Applications for the Synthesis of Bimetallic Cluster Complexes, J. Am. Chem. Soc., V. 124, No. 20, 5628-9, May 2002).
There is a great need to develop novel molecules to which cancer cells are sensitive. Towards that end, the present invention describes organo-metallic complexes comprising ruthenium, and their charge transfer properties.
The formation of nucleosomes in which DNA is wound around a histone spool is believed to be a natural form for condensed DNA and gene suppression. For example, in a prior invention, we introduced a palladium-lipoic acid complex (PLA) which produces heterochromatin in tumor cells. PLA is in Phase II clinical trials.
What was demonstrated through PLA studies was that tumor DNA could be condensed by electron reduction. The PLA studies also showed that an electron redox pathway could be catalyzed by the spin properties of a D-orbital metal bonded to a ligand involved in energy metabolism (lipoic acid). After the example of PLA, other molecular systems were sought which were spin active and strategically located in the cell.
It was believed there were other reservoirs of cell charge besides the mitochondrial site I of lipoic acid. For example, membrane charge might conceivably be mobilized. Membrane charge could theoretically become spin activated, and made resonant with DNA or histone. Therefore we sought a structural site capable of electron transfer from the cell membrane. For this reason, membrane sphingomyelin, which contains the phosphocholine dipole along with fatty acid chains, was of interest.